Immune therapy has already been established as a central component of many cancer treatment regimens. Tumors express a wide variety of proteins that can be recognized by the immune system. In addition to mutated proteins and fusion proteins, the immune system can recognize developmentally and tissue-restricted proteins, as well as proteins highly overexpressed in cancer cells. Established therapies employ a variety of manipulations to activate antitumor immunity. These include passive immunization with monoclonal antibodies, introduction of adjuvants into the tumor microenvironment and systemic delivery of cytokines. Immune therapy can ameliorate the toxic effects of standard chemotherapy and is an essential element in the curative mechanism of bone marrow transplantation for hematologic malignancies. To date, experimental cancer immune therapies are based on established treatment regimens with the purpose of devising a more efficacious and less toxic protocol.
Adoptive T Cell Therapy
Adoptive T cell therapy relies on the in-vitro expansion of endogenous, cancer-reactive T cells. These T cells are harvested from cancer patients, manipulated and then reintroduced as a mechanism for generating productive tumor immunity. Adoptive T cell therapy has had some promising early clinical results and has been associated with clinical responses in a minority of patients with metastatic melanoma. CD8+ cytotoxic T lymphocytes are the primary effector cells in adoptive T cell therapy. However, CD4+ T cells may also play an important role in maintaining CD8+ cytotoxic function and transplantation of tumor reactive CD4+ T cells has been associated with some efficacy in metastatic melanoma. T cells used in adoptive therapy can be harvested from a variety of sites, including peripheral blood, malignant effusions, resected lymph nodes and tumor biopsies. Although T cells harvested from the peripheral blood are technically easier to obtain, tumor-infiltrating lymphocytes (TILs) obtained from biopsies may contain a higher frequency of tumor-reactive cells. In practice, obtaining sufficient cells from tumor biopsies is difficult, although this approach has been used successfully in patients with melanoma.
Once harvested, T cells can be expanded either through polyclonal stimulation with activating antibodies or through exposure to specific tumor antigens, however this second approach requires the identification of relevant targets. Given the frequency of antigen loss variants in current clinical trials, the selection of appropriate targets may be challenging, potentially making polyclonal stimulation a more attractive approach.
In addition to antigen-loss variants, adoptive T cell therapies are limited by the replicative potential of cultured T cells. Several strategies, including the enforced expression of costimulatory proteins and telomerase, have been used to attempt to extend the life span of cultured T cells. IL-15 has also been considered as a possible additive to cultures in order to enhance the production of cytotoxic cells. Intriguingly, engraftment of adoptively transferred T cells appears to be enhanced in lymphodepleted hosts and strategies to combine pretreatment with lymphodepleting chemotherapy and adoptive T cell transplantation appear to increase treatment efficacy significantly.
Two alternative approaches attempt to circumvent low levels of endogenous antitumor reactivity in the peripheral blood by directly supplying T cells with the ability to recognize tumors. T cells harvested from the peripheral blood can be engineered to express T cell receptors (TCRs) that have been selected for tumor recognition. This approach has been previously tested in metastatic melanoma. However, because TCR recognition of an antigen is MHC restricted, each engineered TCR can only be used in patients with the required MHC allele. MHC restriction can be bypassed by engineering T cells to express novel chimeric fusion proteins that link the antigen-binding domain of the B cell receptor with the signaling component of the TCR complex. These “T-bodies” can directly bind tumor antigens, leading to T cell activation, but can be used to target only cell surface overexpressed proteins while TCRs recognize peptides derived from proteins in all cell compartments.
TCR Affinity and T Cell Activity
Previous experiments in mouse models and clinical work have led to the widely accepted concept that for successful TCR gene therapy the generation of high-avidity T cells is a prerequisite. In a mouse tumor model it was demonstrated that complete eradication of solid tumors was possible only if the targeted tumor antigen was expressed at high levels so that it could be cross-presented by the surrounding stroma cells. In naturally occurring tumors, the amount of antigen detectable on tumor stroma is likely to be very low, suggesting that highly sensitive T cells are necessary to promote this bystander effect. Furthermore, high-affinity coreceptor-independent TCRs may allow the generation of both cytotoxic and helper T cells to synergize in the anti-tumor effect. The dependency of T cell activity on the affinity of the TCR to its ligand is one of the aspects currently under intensive research. Studies in this topic show, that when engineering higher affinity TCR mutants they retain their responsiveness to peptide-MHC complexes, however there is indeed a limitation to the level of affinity improvement which can be gained when responsiveness is kept. These studies, used artificial methods for the introduction of mutations, as well as predictions for the sites in which to induce mutations i.e. CDR regions. In doing so these studies limit the overall number of mutations generated and also use a rather laborious method for their generation.
An alternative strategy to overcome T cell tolerance is based on the in-vitro affinity maturation of TCRs isolated from low avidity T cells. In this scenario, TCRs are subjected to in-vitro mutagenesis followed by selection of TCR sequences with improved binding affinity for the specific MHC/peptide combination. This affinity-maturation is achieved by using TCR display libraries expressed in mammalian cells, yeast or on the surface of a phage. This elegant strategy can be used to convert low avidity TCRs, isolated from a repertoire affected by tolerance, to high affinity receptors that are not present in the natural repertoire. The development of tetramer technology has made it easier to isolate T cells specific for particular antigens for example, CMV and it is now possible for direct infusion of these highly purified, specific CD8+ T cells from transplant donors to take place within just four hours of selection. Although T cell immunotherapy against viral targets has proven to be very successful, it is less straightforward when it comes to tumor-associated antigens (TAA)s. TAAs are inherently less immunogenic than viral antigens and cancer patients are usually immunocompromised either by the disease itself or by the treatment they are receiving. This emphasizes a need for high degrees of maturation of the TCR in its target recognition areas.
Somatic Hypermutation (SHM)
There are times when the requirements for biological function exceed the information content of the genome. B-lymphocytes of the adaptive immune system face just such a dilemma: how to extract a virtually infinite repertoire of antigen (Ag)-recognition from a finite supply of genomic information. A “one gene to one Ag” library is unfeasible, so instead, repertoire diversity is achieved through somatic alterations of the immunoglobulin (Ig) locus, which encodes the cell surface receptor responsible for antigen recognition. Ig molecules contain two light chains and two heavy chains arranged in a roughly Y-shaped configuration. The N-terminal prongs (Variable, or V region) specify the Ag-recognizing capacity, whereas the C-terminal stem (Constant, or C region) specifies the effector functions of the molecule. Somatic diversification events occur at both portions of the Ig. Primary diversification occurs in early B cell development during assembly of the V region, a process called V(D)J recombination. This involves the joining of three segments Variable (V), Diversity (D) and Joining (J) randomly selected from a germline pool of multiple gene segments. A mature B lymphocyte which has undergone V(D)J recombination is then distinct from all others at three levels: (i) the choice of V, D, and J segments, (ii) the combination of rearranged heavy and light chains and (iii) junctional insertions and deletions which occur during rearrangement. Somatic hypermutation (SHM) introduces point mutations into the V region antigen-binding pocket, creating Ig variants with enhanced affinity for a particular Ag. These mutations arise at a rate of 10−3/basepair/generation, several orders of magnitude above the rate of spontaneous mutation.
Activation Induced Cytidine Deaminase (AID)
For four decades after it was first proposed that genetic diversity of the precursors of antibody-forming cells arises from a high rate of spontaneous mutation, the identity of the mutator remained unknown. It wasn't until 1999 that Honjo and colleagues identified Activation-Induced Cytidine Deaminase (AID) as the key factor that triggers not only SHM but also CSR (Muramatsu et al., 2000; Muramatsu et al., 1999). Gene conversion was later shown to be dependent on AID, implicating AID as a fundamental mediator of the Ig diversification processes. Based on sequence homology, AID was classified into the APOBEC family of polynucleotide Cytidine deaminases, which perform hydrolytic deamination of Cytidine (C) to Uridine (U). Much like its APOBEC relatives, AID contains a canonical Cytidine Deaminase motif, with key Histidine and Cysteine residues used for zinc coordination and catalysis. The positively charged N terminus carries a putative bipartite nuclear localization signal, though its nuclear localization capacity has not been definitively shown. The C terminus, in contrast, harbors a Leucine rich nuclear export signal, which accounts for the predominantly cytoplasmic distribution of the AID protein. Noting the homology between AID and the well characterized RNA-deaminase APOBEC1, the original discoverers of AID proposed that it edited and activated the mRNA of a SHM- or CSR-catalyzing factor.
PCT Pub. No. WO 03/061363 provides methods for causing mutations in genes expressed in eukaryotic cells; the methods involve expressing AID in the cells. Also provided are cells expressing AID.
PCT Pub. No. WO 06/053021 provides methods using SHM for producing polypeptide and nucleic acid variants.
PCT Pub. No. WO 10/132,092 provides fusion molecules comprising a cytidine deaminase polypeptide and a single stranded DNA binding protein. The '092 publication further provides methods of using the fusion molecules to induce mutations in target genes or polynucleotide sequences.
U.S. Pat. No. 7,569,357 provides T cell receptors (TCRs) that have higher affinity for a ligand than wild type TCRs. These high affinity TCRs are formed by mutagenizing a T cell receptor protein coding sequence to generate a variegated population of mutants of the T cell receptor protein coding sequence; transforming the T cell receptor mutant coding sequence into yeast cells; inducing expression of the T cell receptor mutant coding sequence on the surface of yeast cells; and selecting those cells expressing T cell receptor mutants that have higher affinity for the peptide/MHC ligand than the wild type T cell receptor protein.
U.S. Pat. No. 7,608,410 provides a method of increasing the affinity and/or decreasing the off-rate of a given TCR specific for a given target pMHC.
None of the background art, however, discloses or suggests increasing the affinity of a TCR to its ligand by subjecting the TCR gene to somatic hypermutation, particularly using the mutator enzyme AID. Further, none of the background art discloses or suggests that the affinity maturated TCRs may be used to create anti-tumor reactive T cells.
There exists a long-felt need for more effective means for generating T cells that bear TCR with high functional avidity that have the capacity to recognize their MHC-peptide ligands on pathogenic agents, including but not limited to, tumor cells. There is a further need for methods for the rapid and effective generation of antigen specific T cells which can be used in adoptive cell transfer. Furthermore, there is a need for T cell based pharmaceutical compositions that can be used for treating a patient suffering from a disease, including but not limited to cancer.